RIP3 induces ischemic neuronal DNA degradation and programmed necrosis in rat via AIF

We have reported that nuclear translocation of Receptor-interacting protein 3 (RIP3) involves in neuronal programmed necrosis after 20-min global cerebral ischemia/reperfusion (I/R) injury. Herein, the underlying mechanisms and the nuclear role of RIP3 were investigated further. The necroptosis inhibitor necrostatin-1 (Nec-1), the autophagy inhibitor 3-methyladenine (3-MA), and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-al (Ac-DMQD-CHO) were administered intracerebroventricularly 1 h before ischemia. Protein expression, location and interaction was determined by western blot, immunofluorescence or immunoprecipitation. Most CA1 neuronal death induced by 20-min global cerebral I/R injury was TUNEL-positive. Neuronal death and rat mortality rates were greatly inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. And no activation of caspase-3 was detected after I/R injury. Caspase-8 was expressed richly in GFAP-positive astrocytes and Iba-1-positive microglia, but was not detected in Neun-positive neurons. The nuclear translocation and co-localization of RIP3 and AIF, and their interaction were detected after I/R injury. These processes were inhibited by Nec-1 and 3-MA pre-treatment, but not by Ac-DMQD-CHO. The formation of an RIP3-AIF complex and its nuclear translocation are critical to ischemic neuronal DNA degradation and programmed necrosis. Neurons are more likely to enter the programmed necrosis signal pathway for the loss of caspase-8 suppression.